Recently, it has been reported that a circulating tumor cell (CTC), circulating endothelial cell (CEC) or bone marrow derived-circulating endothelial progenitor (CEP) is useful as a biomarker for estimating an effect of chemotherapy (NON-PATENT LITERATURE 1). The detection of the circulating tumor cell (CTC) has the following advantageous effects. That is to say, it is useful for detecting a metastatic cancer or treating cancer to detect the circulating cancer cells in blood, for the following reasons.
(1) A cancer metastasis means that cancer cells detached from cancer tissues migrate to peripheral blood and proliferate in other tissues reached.
(2) It is reported that the number of CTCs correlates with cancer metastasis and prognosis.
(3) Clinically, a degree of progression, degree of malignancy, and prognosis of breast cancer are diagnosed by the number of CTCs.
When such cancer cells are detected in peripheral blood of a cancer patient, it is necessary to detect very low concentrated cells which are approximately one billionth of the high concentrated blood cells. Therefore, a count loss of cancer cells or a cross-contamination between samples of patients leads to a serious misdiagnosis. As an example of the measurement of CTCs, a Cell Search System (U.S.A), which is only a commercially-available product, is used. A method used in the Cell Search System is disclosed in PATENT LITERATURE 1 and NON-PATENT LITERATURE 1. In particular, cells are subjected to nuclear staining and cytokeratin staining, and react with CD326 antibody immobilized-magnetic beads. The cells are surfaced by a magnetic field, and scanned with a laser beam to obtain fluorescent imaging of the cell. Then, someone decides whether or not the cell is CTC from the fluorescent imaging, and thus the judgment is not an automatic one by an apparatus. Further, there is a method called CTC-chip. The method is disclosed in PATENT LITERATURE 2. In particular, a chip, wherein 80,000 micro posts are formed on a silicon wafer the size of a business card, is used. These 80,000 micro posts are immobilized with an anti-CD326 antibody. Blood is applied to the chip, and then the number of CTCs is measured by recognizing all images of the micro posts. PATENT LITERATURE 3 discloses the following method. Specifically, the CTCs detected by the method disclosed in PATENT LITERATURE 1 are collected, and then the CTCs are genetically analyzed by a fluorescence in situ hybridization (FISH). Further, NON-PATENT LITERATURE 2 discloses a method using a virus with which cancer cells can be specifically infected. The cancer cells are infected with the virus to thereby express a fluorescence protein i.e. the GFP and the cancer cells are detected by a fluorescence microscope. Even further, NON-PATENT LITERATURE 4 describes a method for isolating specific cells from cell population containing many types of cells. In particular, antigen-antibody reactions are carried out by bringing multiple types of thermoresponsive magnetic particles which are immobilized with different types of antibodies respectively, into contact with many types of cell populations. Then, based on the antigen-antibody reactions, many types of cell populations are selectively separated by serially carrying out separations using a gradient field at various temperatures. The numbers of the separated cells are measured by a flow cytometer having a flow cell capable of collecting a sample liquid, and then the cells are collected.
In addition, as a basic research reagent kit for magnetically concentrating CTCs in peripheral blood, the CD326 (EpCAM) Tumor Cell Enrichment and Detection Kit (Miltenyi Biotec, Catalog #130-090-500) is known. This kit is composed of magnetic beads immobilized with CD326(EpCAM) antibody, a fluorescence labeled anti-cytokeratin antibody, and the like. In the kit, the fluorescence of cytokeratin staining must be detected. Therefore, all the concentrated cells are dead by staining intracellular cytokeratin. Further, a reagent kit for magnetically concentrating epithelial cells, i.e. the HUMAN EpCAM POSITIVE SELECTION KIT (Stem Cell Technologies, Catalog #18356) is also known. However, a protocol for concentrating CTCs in blood is not described in its catalog. Further, a reagent kit (Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Catalog #15167)) for negatively selecting CTCs in blood is also known. In the reagent kit, erythrocytes and leucocytes, which are not intended cells, are cross-linked using antibodies and are subjected to a density gradient centrifugation thereof. However, there is no demonstrable indication that the cells concentrated by the reagent kit are only CTCs. Further, a reagent kit (Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Catalog #14152)) for concentrating CTCs by negative selection using antibody-immobilized magnetic beads and a magnet, is also known. However, there is no demonstrable indication that the cells concentrated by the reagent kit are only CTCs. In magnetic beads immobilized with an antibody against a fluorescence molecule, i.e. allophycocyanin (hereinafter referred to as “APC”) (APC Selection Kit (Stem Cell Technologies, Catalog #18451)), APC is labeled with an antibody, and then a magnetic bead is bound to APC. Further, PATENT LITERATURE 5 discloses a technology of a conventional flow cytometer in which a liquid-feeding system containing a flow cell is fixed.